The present invention relates to fluorometric assays and, more particularly, to improved fluorogenic compounds for use in assays for the determination of peroxidase or peroxide.
Zaitsu and Ohkura, Analytical Biochemistry, 109, 109-113 (1980) describe the use of Phenolic compounds as fluorogenic substrates for the horseradish-peroxidase (HRP)-mediated reaction with hydrogen peroxide in order to assay for peroxidase activity or the peroxide. Of the fluorogenic substrates, 3-(p-hydroxyphenyl)propionic acid (HPPA) was identified as being preferred in providing a rapid and sensitive assay. Tuuminen, et al., Journal of Immunoassay, 12(1), 29-46 (1991) recognized the observations of Zaitsu and Ohkura and applied the use of HPPA as a fluorogenic substrate of labelled HRP in an immunoassay.
A limitation accompanying the use of HPPA as a fluorogenic substrate for peroxidases used as labelling enzymes in enzyme immunoassay methods was recognized in Japanese Patent Application No. 4-234998 filed on Dec. 27, 1990, by Kohusai Shiyaku K. K. It was observed that peroxidase-mediated enzyme immunoassay methods using HPPA were intrinsically highly sensitive, but that HPPA formulated in buffers to provide the substrate solution undergoes condensation as a result of the presence of metal ions and is converted into a fluorescent substance which results in a rise in the reagent blank, thus decreasing sensitivity and measurement precision. The Japanese patent application discloses that a chelating agent such as an aminopolycarboxylic acid or aminopolyphosphonic acid, or salts thereof, can be used to stabilize formulated HPPA substrate solutions by reducing the rise in the reagent blank.
While demonstrating an improvement in the assay following the technique illustrated in the Japanese application and other cited literature, there is still room for improvement in the assay. With commercially available fluorogenic phenolic material, or such material purified by conventional techniques, for use as a primary material in formulating a fluorogenic peroxidase substrate, heretofor unrecognized limitations are imposed with respect to the substrate""s utility. One limitation results because of the presence of superoxidation product(s) (as distinguished from oxidative formation of the fluorescent product of HPPA) in the primary material. The superoxidation product(s) have now been discovered as being detrimental because they possess interfering optical qualities which decrease the fluorescent response via an absorptive mechanism (i.e. a filtering effect) within the excitation/emission spectrum of the fluorescent product. This decreases the sensitivity of the substrate when used subsequently in assays. Purification of commercially available material by conventional recrystallization techniques does not result in reduction in the above mentioned superoxidation products.
Additionally, it is has not been heretofore recognized that the background fluorescence of the starting material can be influenced by the manner in which the material is purified; it being assumed that background fluorescence is strictly an intrinsic property of the starting material Accordingly, conventional methods of purification of starting material have not addressed the removal of oxidative products in the starting material which give rise to background fluorescence. Accordingly, low signal to noise ratios accompany the use of such starting materials. And, consequently, both primary materials (before or after conventional purification) that are generally accepted for preparing substrate solutions result in non-optimum assays with respect to high background (decreased signal to noise ratio) and/or decreased sensitivity.
Now in accordance with the present invention, there is provided means for preparing improved fluorogenic phenolic compounds for use as primary materials in preparing substrates with fluorogenic activity in peroxide-mediated assays of peroxidase activity or peroxidase-mediated assays of peroxide. The method provides the phenolic compound in an optically enhanced condition by controlling the presence of superoxidation products while concurrently yielding primary material for use in formulating substrate solutions which have low background fluorescence. The method involves forming under anoxic conditions, i.e. decreased oxygen concentration, a solution which contains a fluorogenic phenolic compound and an aminopolycarboxylic acid or aminopolyphosphonic acid, or salt, thereof, metal chelating agent and, while the solution is maintained under anoxic conditions, recovering the compound from the solution, by for example, crystallization. As so recovered, the compound is in an optically enhanced condition.
The method disclosed herein is contrary to conventional wisdom which teaches against the addition of extraneous substances in crystallization procedures. The method disclosed herein is also contrary to conventional wisdom which instructs the use of activated carbon for removal of optical impurities; the use of which is detrimental to phenolic compounds prepared for use as primary materials for subsequent use in formulating fluorogenic peroxidase substrates.
In further keeping with the present invention another aspect thereof provides a composition of matter with improved optical performance characteristics for use in formulating a fluorogenic substrate solution in peroxide-mediated assays of peroxidase activity or peroxidase-mediated assays of peroxide. The composition contains a fluorogenic phenolic compound in crystal form and a trace quantity of an aminopolycarboxylic acid or aminopolyphosphonic acid, or salt thereof, metal chelating agent, which copurifies with the fluorogenic phenolic compound and which can also arise from the admixture of the metal chelating agent with the compound during washing procedures. Typically, such agents would be present in amount of less than 0.3% by weight, but higher concentrations are not necessarily detrimental since the chelating agent is generally included in the substrate solution from which the phenolic compound is prepared.
The composition of matter above described can be further characterized as having an optical quotient of less than about 1.1 and preferably about 1.0. As used herein, the optical quotient is defined as the ratio of the relative fluorescence (RFU) of a 5 mg/ml concentration of the compound, dissolved in 0.3 M glycine, 2 mM EDTA, pH 10.5 buffer, as compared to the relative fluorescence of a 10 mg/ml concentration of the compound when dissolved in the same buffer, both fluorescent measurements being made after approximately the same elapsed time once the fluorescence intensities of the two solutions have stabilized and under the substantially the same conditions. Having an optical quotient, as above identified, defines the absence of products detrimental to the optical qualities of the fluorogenic phenolic primary material. The detrimental products provide a filtering effect which results in a decrease in fluorescence as concentration increases, and a corresponding increase in optical quotient.
The above described fluorogenic material has reduced intrinsic background fluorescence which is otherwise associated with material not prepared in accordance with the present invention. In this respect, background fluorescence of the fluorogenic phenolic material of the present invention is reduced by at least 3% as compared with the background fluorescence of the parent material from which it is purified, even where the parent material is considered to be highly qualified for use as a primary material.